Recent advances in treating metastatic melanoma, particularly with immune checkpoint inhibitors, have resulted in improved overall survival. Despite this, the mortality from melanoma continues to rise due to the increased incidence and to primary and acquired resistance to immunotherapy. In previous studies we showed that the secreted flavoprotein Renalase (RNLS) is upregulated in melanoma cells and high expression in human tumors is associated with tumor progression and decreased survival. Knock-down of RNLS by RNLS antibodies and an inhibitory peptide in murine models inhibited tumor growth, decreased signaling through the MAPK and PI3K pathways in tumor cells, and caused a marked reduction in CD163+ Tumor Associated Macrophages (TAMs). Furthermore, we showed that increased RNLS production by CD163+ TAMs facilitates melanoma growth by activating STAT3, suggesting that inhibition of RNLS might be a useful approach for treating melanoma by direct tumor cell targeting and by inhibition of CD163+ TAMs. The latter mechanism is particularly interesting as CD163+ TAMs are known to facilitate resistance to tumor cell kill by effector T cells. We propose to test the hypothesis that dysregulated RNLS expression and signaling in melanomas modulates tumor growth and spread and will further study the mechanism thereof. Furthermore, we surmise that RNLS in tumor cells and TAMs modulates both the innate and adaptive immune response to promote cancer growth and spread. Therefore, co-inhibition of RNLS and PD-1 or CTLA4 in CD8+ T cells could result in enhanced anti-tumor activity compared to either approach alone. We will capitalize on unique resources available to our team that include novel animal models, availability of a novel RNLS inhibitor, and access to melanoma specimens from patients treated with immune checkpoint inhibitors, and the studies will be conducted by an interdisciplinary team of basic, clinical and translational researchers. We propose three inter-related but not inter-dependent aims. In Aim 1 we will determine the mechanism by which RNLS inhibition affects signaling pathways within melanoma cells and the mechanism by which RNLS modulates the innate and adaptive immune response to melanoma in vitro and in vivo. In Aim 2 we will fully characterize expression of RNLS in tumor cells characterized for BRAF and NRAS mutations, TAMs (CD163+ or CD86+), CD4 and CD8+ cells in large cohorts of metastatic melanoma tumors, including tumors from patients treated with immune checkpoint inhibitors. In Aim 3 we will test the hypothesis that combination therapy with anti-RNLS antibodies and anti-CTLA4 or anti-PD-1 are synergistic in immune competent murine melanoma models with genetic aberrations relevant to human tumors (mutations in BRaf or NRas). The potential impact of these studies is high ? we are developing a RNLS inhibitor for clinical use. If successful, these studies will lead to future clinical trials of the combination anti-RNLS with an immune checkpoint inhibitor. This approach can be studied in other diseases as well and can thus have far-reaching implications for cancer therapy.